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a novel rice ERF gene, up-regulates ethylene-responsive genes ...

a novel rice ERF gene, up-regulates ethylene-responsive genes ...

1722 Figure 3.

1722 Figure 3. Semi-quantitative RT-PCR analyses of OsERF1 expression in different tissues (A, B) or under different treatments (C, D). Tubulin A was used as a constitutive control. (A) Expression profiles in roots (Rad), leaves (Lea), inflorescences (Inf) and buds (Bud). (B) Expression profiles in developing flowers at male meiosis (Mei), tetrad spore (Tet), uninucleate pollen (Uni), binucleate pollen (Bin) and trinucleate pollen (Pol) stages. (C) Response of OsERF1 expression to low temperature (8 1C) (Tem), 20% PEG (PEG), Eth (0.1 mM ethrel) and ABA (0.01 mM ABA); Con: control. The treatment time was 4 h. (D). Response of OsERF1 expression to 1 mM ethrel under 1 h (Et1), 2 h (Et2), 4 h (Et4) inducement or MeJA (0.1 mM), SA (1 mM) for 4 h; Con: control. Overexpression of OsERF1 in Arabidopsis causes similar phenotypes as in transgenic plants that overexpress known ethyleneresponsive factors The overexpression construct of OsERF1 and the pCAMBIA1302 vector (control) were introduced into Arabidopsis. Among the 25 identified T1 transgenic lines of OsERF1, 11 (44%) lines showed dwarf size, dark-green leaves and delayed bolting, and subsequently produced less siliques (L1, L2, L3, L4, L6 produced less seeds and L5 produced few seeds) or no siliques at all (the other five lines); whereas the remainder (such as L11) had no visible aberrances as compared with wild-type plants. In addition, we identified 17 positive lines transformed with empty vector (pCAMBIA1302) and none of them showed aberrant phenotypes, which implied that the aberrant phenotypes were attributable to OsERF1. DNA gel blot revealed two copies of the transgene had been inserted in L1 and L2, one in ARTICLE IN PRESS Y. Hu et al. Figure 4. Detection of OsERF1 expression in transgenic Arabidopsis plants by RNA gel blot. Total RNAs were prepared from transgenic plants L2, L3, L5, L6, L11 and wild-type controls (WT). rRNAs were used as a marker to indicate that equal amount of RNAs were loaded in each lane. L3, L4 and L6, three in L11, but none in wild type; and the integration sites of the transgene in these lines were distinct from each other. RNA gel blot (Figure 4) revealed that OsERF1 expressed at the highest level in L3, to a slightly lesser extent in L2 and L6, and at a relatively low level in L5. No expression was detected in L11. The difference in expression levels paralleled the deviation in phenotypes among these lines: L3 had the most severe aberrant phenotypes, whereas L11 had no observable aberrant phenotypes, which suggested that the transgene was silenced in L11. Moreover, the presence of nucleus-localized OsERF1 in these overexpressed OsERF1 lines indicated successful translation of the OsERF1 protein (Figure 2). Together, these facts demonstrate that overexpression of OsERF1 results in the aberrant phenotypes. Transgenic plants showed dwarf size as compared with wild-type plants, the mean height of L2, L3 and L6 was about 74% of wild type (Figure 5) and their mean bolting time was delayed about 30 d (Figure 6). In addition, the uppermost flowers (usually the first 10) on the inflorescences of the transgenic plants were infertile (Figure 7E). Detailed observation showed that their gynoecium protruded while their pollens were still immature (Figure 7A and B), but their pollens were viable at mature pollen stage (Figure 7C and D). These aberrant phenotypes are very much alike to those of Arabidopsis plants that overexpress ERF1 or EIN3, which show constitutive ethylene response (Chao et al., 1997; Solano et al., 1998). It is probable that like ERF1, OsERF1 may be involved in ethylene signaling pathway. OsERF1 can activate expression of ethyleneresponsive genes To identify this speculation, we first observed the morphologic features of dark-grown OsERF1-overexpressed seedlings of L6. As shown in Figure 8A, transgenic seedlings showed short hypocotyls and

ARTICLE IN PRESS A novel ERF gene of rice 1723 Figure 5. Overall comparison of wild-type and OsERF1-overexpressed plants L6. (A) Morphology of OsERF1overexpressed plants (right) and wild type (left) at vegetative growth stage. (B). Morphology of OsERF1-overexpressed plants (right) and wild type (left) at reproductive growth stage. height (cm) 40 35 30 25 20 15 10 5 0 plant size bolting mean time 0 WT L2 L3 L6 WT L2 L3 L6 Figure 6. Diagrams of plant size and mean bolting time of wild-type and OsERF1-overexpressed Arabidopsis. (A) A diagram to show mean height of wild-type and OsERF1-overexpressed Arabidopsis (L2, L3, L6). (B) A diagram to show mean bolting time of wild-type and OsERF1-overexpressed Arabidopsis (L2, L3, L6). Figure 7. Comparison of flowers from wild-type and OsERF1-overexpressed plants L6. (A). Wild-type flower (left) and OsERF1-overexpressed infertile flower (right). (B) Dissection structure of wild-type flower (left) and OsERF1overexpressed infertile flower (right). Four sepals and two petals had been stripped off to show the small petals, small and short stamens in OsERF1-overexpressed infertile flower. (C, D) Bright-field micrographs of Alexander-stained anthers from wild-type flower (C) and OsERF1-overexpressed infertile flower (D). Scale bar ¼ 100 mm. (E) Inflorescences and siliques from wild-type (left) and OsERF1-overexpressed plants (right). The magnified part shows OsERF1overexpressed infertile flower. time (day) 100 80 60 40 20

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